Recombinant vectors based on adeno-associated virus (AAV) can efficiently transduce many different cell types, including cells of the retina, resulting in stable gene expression. A major shortcoming of this vector is its small packaging capacity. A trans-splicing approach, which reconstitutes gene expression from two independent AAV vectors, can be used to overcome the vector's packaging limitations. The efficiency of this system to date has been disappointing, and therefore its utility for therapeutic application limited. We demonstrate here that efficiency and cellular specificity of trans-splicing is dependent on selection of the appropriate AAV serotype. Efficiency of transgene expression resulting from trans-splicing in skeletal muscle approaches that obtained when delivering the intact transgene when using AAV2 vectors packaged with AAV5 capsids (AAV2/5). This expands the potential of AAV vectors for retinal gene therapy. The use of AAV2/5 also increases the efficiency of trans-splicing in photoreceptors. Selection of the appropriate AAV serotype is likely to affect efficiency of trans-splicing in other organ systems as well.